Microarrays have become widely applied in proteomic and genomic analysis, as they permit multiplexed analysis of multiple analytes. See, e.g., Ramsay, Nat. Biotechnol. 16, 40-44 (1998); P. Brown, D. Botstein, Nat. Genet. 21, 33-37 (1999); D. Duggan, M. Bittner, Y. Chen, P. Meltzer, J. M. Trent, Nat. Genet. 21, 10-14 (1999); R. Lipshutz, S. P. A. Fodor, T. R. Gingeras, D. J. Lockhart, Nat. Genet. 21, 20-24 (1999). In a microarray, binding agents such as antibodies and/or oligonucleotides are spotted on planar substrates. These binding agents are then contacted with samples including complementary ligands (proteins or complementary oligonucleotides, as applicable) and permitted to bind or hybridize. The binding or hybridization is then detected. Because either the identity of the binding agents or the complementary ligands are known, by tracing their identity in the array, the complementary oligonucleotides or proteins can be determined. This is an effective method for identification or quantification of analytes in a sample.
The principal techniques of oligonucleotide array fabrication include: spotted arrays, and refinements of the original “spotting” in the form of pin transfer or ink jet printing of small aliquots of probe solution onto various substrates, as illustrated in V. G. Cheung, et al., Nat. Genet. 21, 15-19 (1999); sequential electrophoretic deposition of binding agents in individually electrically addressable substrate regions, as illustrated in J. Cheng, et al., Nat. Biotechnol. , 541-546 (1998); and methods facilitating spatially resolved in-situ synthesis of oligonucleotides, as illustrated in U. Maskos, E. M. Southern, Nucleic Acids Res. 20, 1679-1684 (1992); S. P. A. Fodor, et al., Science 251, 767-773 (1991) or copolymerization of oligonucleotides, as illustrated in A. V. Vasiliskov, et al., BioTechniques 27, 592-606 (1999). These techniques produce spatially encoded arrays in which the position within the array indicates the chemical identity of any constituent probe.
The reproducible fabrication of customized arrays by these techniques requires the control of microfluidics and/or photochemical manipulations of considerable complexity to ensure consistent performance in quantitative assays. Microfluidic spotting to produce, in quantitatively reproducible fashion, deposited features of 100 μm diameter involves dispensing of nanoliter aliquots with tight volume control, a task that exceeds the capabilities of currently available fluid handling methodologies. In addition, exposure of binding agents to air during the deposition process, typically several hours' in duration, has uncontrollable impact on the molecular configuration and the accessibility of the binding agents in subsequent binding assays. In-situ array synthesis relies on a sequence of multiple masking and photochemical reaction steps which must be redesigned to accommodate any changes in array composition. Finally, assay performance must be assessed “in-situ” for each array subsequent to immobilization of binding agents, an aspect of array manufacturing which raises difficult quality control and implementation issues.
As an alternative to solve many of the problems associated with spotted arrays, microbead particles bound to oligonucleotide probes have been used. See U.S. application Ser. No. 10/271,602, “Multiplexed Analysis of Polymorphic Loci by Concurrent Interrogation and Enzyme-Mediated Detection” filed Oct. 15, 2002; Ser. No. 10/204,799 “Multianalyte Molecular Analysis Using Application-Specific Random Particle Arrays,” filed on Aug. 23, 2002, both being incorporated by reference. The beads are deposited on a substrate, and preferably affixed thereto, to form an array. Among the principal advantages are that the beads are encoded so that particular probes associated with particular beads can be determined by decoding. This obviates the need, associated with spotted arrays, to form arrays with particular probes in particular positions (spatial encoding).
Affixing the beads to the substrate is desirable because if they move about on the substrate, decoding signals cannot be localized and accurately interpreted. One of the advantages of a bead array is that the signal from the array and the decoding can be accomplished using an ordinary microscope. For example, a microscope can acquire a fluorescent signal from bound ligands in the array or can detect color differences which encode the beads. The array should be sized such that the entire array can be viewed in a single field, i.e., all at once, under the microscope.
One method of immobilizing beads involves confining them to wells in the substrate, where the wells are size-matched in diameter to the beads (i.e., only slightly larger than the beads). The height of the wells is preferably also about the same as that of the beads. FIG. 1 shows a cross-sectional view of a collection of beads in wells that function as mechanical traps. As long as the substrate faces upwardly, gravity inhibits the beads from escaping from the wells. However, forces produced by fluid transport, for example the combination of lateral and normal forces generated by the movement of an air-liquid interface over the substrate, can readily dislodge beads which are not adhered to the substrate. What is needed are compositions and methods to overcome such forces and permit consistent manufacture of arrays of immobilized beads.
By way of background, it is noted that the magnitude of the adhesion force between two solid surfaces depends on a number of factors, including surface chemistry, relative humidity, temperature, surface roughness, time of contact, nature of the material, and others. However, for two surfaces to establish adhesive contact, they must first approach each other closely. A number of colloidal forces control and modulate the approach of two surfaces. FIG. 2 shows the dependence of interaction energy (U) on separation for several commonly encountered forces. Each of these forces can be controlled by changing various parameters. For example, the long-range electrostatic repulsive force between two surfaces bearing charges of similar sign can be controlled by addition of salt, which screens the repulsive force, thereby permitting the two surfaces to come into closer proximity. In absence of screening, the equilibrium “contact” distance for a negatively charged polystyrene microparticle (several microns in diameter) from a charged flat surface like glass is on the order of 100's of nm, because the electrostatic repulsive forces which counteract gravity. Under such circumstances, particles remain sufficiently far from the surface so as to remain substantially unaffected by attractive interactions that operate at short length scales. It is noted, however, that ligand-receptor type interactions (for example, hybridization between complementary oligonucleotides) are attractive in nature and capable of operating at long length scales.
Capillary forces also can be very effective in bringing two surfaces close to each other. For example, the capillary force (Fc) between a rigid sphere of radius R and a flat surface has the functional form Fc˜4πRσ cos θ, where θ is the local contact angle, and σ the interfacial tension of the liquid forming the capillary film. See Israelachvili, J. N. Intermolecular and Surface Forces, Academic Press, New York, 1985. For a wetting film, the large interfacial tension of the liquid leads to a correspondingly large value of Fc.
Once in contact, whether two surfaces will adhere is not solely determined by the net attraction between molecules. Bodies deform macroscopically in response to being placed in contact and hence the bulk contact geometry as well as material properties, such as elastic modulus and hardness, all affect the conformity and adhesion of one surface to another. According to JKR theory (Johnson, K. L, Kendall, K., Roberts, A. D. Surface Energy and the Contact of Elastic Solids. Proc. R. Soc. London A 1971, 324, 301) the variation of the radius of contact ‘a’ under applied load ‘P’ has the form
      a    3    =            R      K        ⁡          [              P        +                  3          ⁢          π          ⁢                                          ⁢          R          ⁢                                          ⁢          W                +                                            6              ⁢                                                          ⁢              π              ⁢                                                          ⁢              R              ⁢                                                          ⁢              W              ⁢                                                          ⁢              P                        +                                          (                                  3                  ⁢                                                                          ⁢                  π                  ⁢                                                                          ⁢                  R                  ⁢                                                                          ⁢                  W                                )                            2                                          ]      where K denotes an elastic constant, W ˜√{square root over (γ1γ2)}, denotes the work of adhesion (where γ1 and γ2 are the surface energies of the two surfaces in contact) and R denotes the radius of curvature. In the absence of any applied load (P), the equilibrium contact radius is:
  a  =            a      0        =          [                        6          ⁢                                          ⁢          π          ⁢                                          ⁢                      R            2                    ⁢          W                K            ]      The theory also predicts an adhesive contact force (Fadh):
      F    adh    =            -              3        2              ⁢    π    ⁢                  ⁢    R    ⁢                  ⁢    W  A large area of contact enhances adhesion, however it alone is not sufficient. For example, a wetting liquid achieves an excellent contact, but does not generate significant adhering force, because it does not resist shear deformation.
In reality, material surfaces are rough and hence are never in intimate contact. If the real area of contact is small, adhesion is weak. For rough surfaces, surface phenomena alone cannot account for the adhesion and many other macroscopic phenomena come into play, including, the degree of surface roughness, the maximal normal force applied to the contact, time for which the surfaces are in contact, bulk molecular structure and dynamics, and others. One of the early studies by Greenwood and Tripp (Greenwood, J. A., Tripp, J. H. The Elastic Contact of Rough Spheres. J. Appl. Mech. 1967 (March) 153-159) concluded that the effective initial contact area between rough surfaces is given by:α=√{square root over (2Rσroughness)}where σroughness is the root mean square of the surface asperities and R the bulk radius of curvature of the system (radius of the sphere for a sphere and flat system). A recent study by Quon et al. (Quon, R. A., Knarr, R. F., and Vanderlick, T. K. Measurement of the Deformation and Adhesion of Rough Solids in Contact. J. Phys. Chem. B 1999, 103, 5320-5327) concluded that systems that are in initial contact over an area with a radius greater than that predicted above are strongly adherent and ones with a smaller radius of contact are either weakly adherent or non-adherent. Thus, for rough solids, the adhesion improves with increasing initial loads. Under the influence of an increased initial load, asperities deform, permitting the surfaces to approach more closely and thereby permitting van der Waals attractions to contribute to adhesion.
In immobilizing bead arrays on silicon substrates (chips) for multiplexed analysis of multiple analytes, the immobilization must take place after completion of the array assembly process. Also, the immobilization protocol should not in any substantial manner affect the on-chip bioassays, i.e., it should, instead, preserve receptor moieties displayed on bead surfaces, leave binding kinetics unaffected and minimize non-specific binding.